1. Field of the Invention
This patent application relates to hybrid and/or single-chain rare-cutting endonucleases, called meganucleases, which recognize and cleave a specific nucleotide sequence, to polynucleotide sequences encoding for said rare-cutting endonucleases, to a vector comprising one of said polynucleotide sequences, to a cell or animal comprising one of said polynucleotide sequences or said rare-cutting endonucleases, to a process for producing one of said rare-cutting endonucleases and any use of the disclosed products and methods. More particularly, this invention contemplates any use of such rare-cutting endonuclease for genetic engineering and gene therapy.
2. Brief Description of the Prior Art
Meganucleases constitute a family of very rare-cutting endonucleases. It was first characterised at the beginning of the Nineties by the use (in vivo) of the protein I-Sce I (Omega nuclease, originally encoded by a mitochondrial group I intron of the yeast Saccharomyces cerevisice). Homing endonucleases encoded by introns ORF, independent genes or intervening sequences (inteins) are defined now as “meganucleases”, with striking structural and functional properties that distinguish them from “classical” restriction enzymes (generally from bacterial system R/MII). They have recognition sequences that span 12-40 bp of DNA, whereas “classical” restriction enzymes recognise much shorter stretches of DNA, in the 3-8 bp range (up to 12 bp for rare-cutter). Therefore, the meganucleases present a very low frequency of cleavage, even in the human genome.
Furthermore, general asymmetry of Meganucleases target sequences contrasts with the characteristic dyad symmetry of most restriction enzyme recognition sites. Several Meganucleases encoded by introns ORF or inteins have been shown to promote the homing of their respective genetic elements into allelic intronless or inteinless sites. By making a site-specific double-strand break in the intronless or inteinless alleles, these nucleases create recombinogenic ends, which engage in a gene conversion process that duplicates the coding sequence and leads to the insertion of an intron or an intervening sequence at the DNA level.
Meganucleases fall into 4 separated families on the basis of pretty well conserved amino acids motifs. One of them is the dodecapeptide family (dodecamer, DOD, D1-D2, LAGLI-DADG, P1-P2). This is the largest family of proteins clustered by their most general conserved sequence motif: one or two copies (vast majority) of a twelve-residue sequence: the di-dodecapeptide. Meganucleases with one dodecapetide (D) are around 20 kDa in molecular mass and act as homodimer. Those with two copies (DD) range from 25 kDa (230 AA) to 50 kDa (HO, 545 AA) with 70 to 150 residues between each motif and act as monomer. Cleavage is inside the recognition site, leaving 4 nt staggered cut with 3′OH overhangs. I-Ceu I, and I-Cre I illustrate the meganucleases with one Dodecapeptide motif (mono-dodecapeptide). I-Dmo I, I-Sce I, PI-Pfu I and PI-Sce I illustrate meganucleases with two Dodecapeptide motifs.
Goguel et al (Mol. Cell. Biol., 1992, 12, 696-705) shows by switching experiments of RNA maturase and meganuclease of yeast mitochondria that the meganuclease badly tolerates sequence switching and loses its endonuclease activity.
Endonucleases are requisite enzymes for today's advanced gene engineering techniques, notably for cloning and analyzing genes. Meganucleases are very interesting as rare-cutter endonucleases because they have a very low recognition and cleavage frequency in large genome due to the size of their recognition site. Therefore, the meganucleases are used for molecular biology and for genetic engineering, more particularly according to the methods described in WO 96/14408, U.S. Pat. No. 5,830,729, WO 00/46385, and WO 00/46386.
Up to now, in a first approach for generating new endonuclease, some chimeric restriction enzymes have been prepared through hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme Fok I (Smith et al, 2000, Nucleic Acids Res, 28, 3361-9; Kim et al, 1996, Proc Natl Acad Sci USA, 93, 1156-60; Kim & Chandrasegaran, 1994, Proc Natl Acad Sci USA, 91, 883-7; WO 95/09233; WO 94/18313).
An additional approach consisted of an alteration of the recognition domain of EcoRV restriction enzyme in order to change its specificity by site-specific mutagenesis (Wenz et al, 1994, Biochim Biophys Acta, 1219, 73-80).
Despite these efforts, there is still a strong need for new rare-cutting endonucleases with new sequence specificity for the recognition and cleavage.